Project Summary The goal of this proposal is to establish a new paradigm for reverse signaling by GPI-linked (type-A) Ephrins in the developing nervous system. Ephrins are membrane-associated ligands that interact with Eph receptor tyrosine kinases in many regions of the nervous system, and they are frequently upregulated at sites of injury and pathogenic lesions in the brain. Because Ephrins and Eph receptors are membrane-associated proteins, they can induce bi-directional signaling: Ephrins stimulate conventional &#147;forward&#148;signaling by activating Eph receptors, but Eph receptors can also trigger &#147;reverse&#148;signaling by stimulating Ephrin-mediated responses. Reverse signaling by transmembrane (type B) Ephrins has been well characterized, but reverse signaling via type A Ephrins remains poorly understood. Analysis of this process has been hindered by the complexity of the vertebrate nervous system, where multiple Ephrins and Eph receptors are often co-expressed and can interact promiscuously. In contrast, the developing enteric nervous system (ENS) of the Lepidopteran species Manduca sexta provides a model preparation in which only one GPI-linked Ephrin (MsEphrin) and its corresponding receptor (MsEph) are expressed in a cell type-specific manner. Migratory neurons that populate the ENS express MsEphrin, while MsEph receptors are confined to midline muscle cells that are inhibitory to migration, and reverse signaling via MsEphrin regulates neuronal guidance at this boundary. Recent studies have also shown that MsEphrin signaling is transduced by a Src-related kinase (MsSrc) in the migratory neurons, providing the first example of this signaling pathway in vivo. The large size and molecular simplicity of this preparation provides a unique combination of embryonic culture assays, time-lapse imaging, and biochemical assays to investigate the mechanisms of Ephrin-A reverse signaling during normal development. Proteomics-based affinity screens of MsEphrin-interacting proteins have identified the signaling/adapter protein RACK1 as a central component of the reverse signaling pathway, and a receptor protein tyrosine phosphatase (PTP10D) has been identified as a candidate co-receptor. An endogenously expressed ADAM secretase (Kuzbanian) has also been implicated in MsEphrin processing that may terminate reverse signaling, and both PTP10D and Kuzbanian interact with RACK1. The experimental assays unique to the developing ENS can now be used to delineate the role of a specific Src isoform (MsSRc42A) and its downstream effectors (Rho and Rho kinase orthologs) in MsEphrin reverse signaling. These components will then be used to test the role of PTP10D and Kuzbanian in the transduction and termination of MsEphrin activation, and to test the model that a &#147;RACK1 rheostat&#148;integrates the response of migratory neurons to MsEph receptors in the developing ENS. If successful, these experiments will establish a novel paradigm for Ephrin-A reverse signaling in the nervous system. They may also promote new therapeutic strategies targeting misregulated Ephrin-A reverse signaling in a variety of neurological lesions and within invasive glioblastomas.